In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase, or GAA) deficiency.Acid maltase deficiency is fatal in childhood if the enzyme is absent and leads to a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later decades if some enzyme is present. Earlier studies in our laboratory of the most common adult mutation with an in vitro model system have shown that a single base mutation in the polypyrimidine tract towards the end of intron 1 reduces the transcription rate, apparently by altering the binding of a splicing factor, and alters the ratio of splice variants to favor the splicing of non-productive mRNA. Furthermore, a silencer was identified elsewhere in this intron, and was considered a possible target for therapeutic intervention. If the silencer could be overcome, the resulting upregulation of gene transcription might overcome the reduced synthesis in the common adult allele. In the past year, localization of the silencer activity in Hep G2 cells has been completed. The activity lies within a 25 bp region within which there are adjacent sites for two known transcription factors, YY1 and Hes-1. Experiments have shown that both of these factors bind to the region. By mutating the region, it has further been shown that alteration of either site is sufficient to reverse the silencer activity. These studies establish, therefore, that the two transcription factors work cooperatively to down-regulate transcription of the gene. It will now be interesting to see if the silencing can be reversed by a phosphorothioate oligodeoxynucleotide decoy. And it will be important to examine the activity of this stretch in other cell types. The findings so far are intrinsically interesting since so little is known of the control of so-called housekeeping genes. In fact, this is the first example of transcriptional control of a gene in this family localized to an intron. Of even greater interest is the involvement of Hes-1, a target gene in the Notch signaling pathway. Hes-1 is a gene that is critical for nervous system and thymic maturation. Of more practical importance is the recognition that two proteins must act together to silence this gene, implying that either alone might be a target for reversing the silencing activity. In order to provide an animal model for testing several proposed therapies for acid maltase deficiency which are actively underdevelopment in our group and by groups in the Netherlands, in New York, at Duke, and at Johns Hopkins, we have made knockout models of the GAA gene in mice. With the assistance of Dr. Brian Sauer of NIDDK and Dr. Edward Ginns of NIMH, we have created complete knockouts of the gene by the introduction of the neo gene in exon 6 and in exon 14. Homozygous F2 offspring rapidly accumulate glycogen in cardiac and skeletal muscle. Females show impaired performance in muscle testing by quantitative open field observation and by ability to hang on a wire screen or move on a rotating rod, and by several months, they develop a grossly waddling gait. The exon 6 knockout was designed so that the neo gene and the whole of exon 6 could be deleted permanently by mating the mice to mice transgenic for the CRE recombinase. These mice delta 6/delta 6 have a slower onset of clinical disease despite total absence of GAA activity, apparently because of differences in the background genes of the two strains. In the past year we set out to determine whether increased cellular glycogen synthesis brought about by transgenes for the glucose transporter, GLUT 1, or glycogen synthetase (GS) itself could accelerate disease in the knockout mice. Both did accelerate the disease, but the histologic findings in the knockout mice with transgenic GS accumulated not more glycogen but another glucose polymer, polyglucosan. This accumulation appears to occur because of the relative insufficiency of the so-called brancher enzyme which allows the characteristic branched glycogen structure to develop. These findings have implications for understanding an important cause of childhood epilepsy, progressive myoclonus epilepsy (Lafora body disease). Our collaborators in the Department of Pediatrics at Duke have developed a promising vector for the gene therapy of acid maltase deficiency. The efficacy of the vector has now been tested in our knockout mice, and it has been established that enzyme made by vector that has settled in the liver is secreted in sufficient quantity to reverse glycogen accumulation in muscle. Our collaborators in the Department of Pediatrics at Johns Hopkins and at the University of Florida have developed an adeno-associated virus (AAV) vector with the acid maltase gene. Direct infection of the heart or skeletal muscles of our knockout animals led to functional as well as biochemical correction. We are currently developing tetracyline controllable, tissue specific acid maltase transgenes to determine which is the preferable route to prevent and to correct disease in the knockout mice. Because both enzyme replacement and gene-based therapies of this disease will have to be monitored in living human subjects, we have collaborated with the Department of Radiology to seek a detectable magnetic resonance signal for glycogen. It has proved possible to detect glycogen in solution at the concentrations found in muscle from patients with acid maltase deficiency. Furthermore, the signal could be detected in muscle excised from a clinically symptomatic knockout mouse. Unfortunately, the glycogen signal lies on a limb of the water signal, and surrounding tissue water renders it impossible to see the signal in the intact animal. Nevertheless, because it may prove possible to obtain a more localized signal in human limbs, we have begun a study in which areas appearing abnormal whole patient MRI are sampled under CT guidance and will be tested to see if there is a correlation between tissue level of glycogen and the degree of signal abnormality.The members of the group joined with others studying acid maltase deficiency to hold an international workshop on the disease which was held at NIH in December 1998 with about 70 participants from NIH and many laboratories in the United States and a number of foreign countries, including the Netherlands, Japan, Australia, Taiwan, France, and England.